Degenerative retinal diseases resulting from permanent loss of retinal neurons pose a great public health risk as no effective cell replacement therapies are currently available. De novo retinal regeneration from stem cells or retinal precursors could lead to a potential remedy for retinal degeneration diseases. Studies to identify genes and genetic processes essential for the formation of retinal neurons will provide the foundation for retinal regeneration. This proposal aims to reveal the molecular mechanisms governing the generation of retinal ganglion cells (RGCs), a group of neurons degenerated in glaucoma diseased eyes. The long-term objective of this proposal is to understand the role of an Atonal-class basic helix-loop-helix (bHLH) transcription factor, Math5, in the process of retinal neurogenesis and its interactions with other transcription factors in regulating the cell fate choices during retinal development. Knockout studies have shown that loss of Math5 leads to the absence of RGC formation and a concurrent increase in the generation of amacrine and cone photoreceptor cells, implying the dual roles of Math5 in promoting RGC differentiation pathway and inhibiting non-RGC differentiation pathways. Furthermore, we have shown that Math5 positively regulates the expression of RGC- specific transcription factors like Brn3b and Isl1, and negatively regulates non-RGC factors such as Bhlhb5, NeuroD, Math3, and Ngn2. The Aim 1 of this proposal is designed to characterize the role of Bhlhb5 in the development of selective subtypes of amacrine and cone bipolar cells. Lineage analysis using Bhlhb5-Cre (Cre recombinase) mice will be carried out to test if Bhlhb5 expression defines amacrine and cone bipolar subtypes. In Aim 2, the role of Isl1 in retinal genesis will be investigated in retina-specific Isl1 knockout mice. In addition, the synergistic effects of Math5 and Isl1 co-expression in promoting RGC formation from retinal precursors will be assessed by knocking-in Isl1 in Math5 locus. The Aim 3 is proposed to test the regulatory relationship of Math5 and its known downstream target genes. Chromatin immunoprecipitation (ChIP) experiments using anti- HA and genetically modified Math5-HA mice will be used to identify the direct target genes of Math5 as well as to discover novel Math5 target genes. Alternatively, a Tamoxifen-inducible Math5 (Math5ER, a fusion of Math5 and estrogen receptor ligand-binding domain) will be used to identify genes directly activated by Math5 in the presence of Tamoxifen and protein synthesis inhibitor, cycloheximide.